This invention relates to analyses of the kind in which a radioactive element or compound is partitioned between two phases and the proportion of the activity in each phase determined. Typical of such analyses are competitive assays of the kind in which an unknown amount of the compound to be assayed and a standard amount of a radioactively-labelled version of the compound compete for reaction with a standard amount of another reagent. This technique is widely used in the medical field for assaying hormones and other substances, using as the other reagent the antibody in immune systems or some other specific reactor in non-immune or non-hormone systems.
The principle of the technique may be represented by the following scheme: EQU C + C* + R.revreaction. C - R + C* - R
where
C is the compound to be assayed, PA0 C* is the labelled version of the compound PA0 R is the other reagent. PA0 a. drawing liquid from one of the set of vessels into a defined measuring region so that the liquid fills the said region, PA0 b. measuring radiation emitted by the liquid in the region, PA0 c. expelling the liquid from the region,
The amount of R is arranged to be insufficient to react with all of C + C*. As the reaction is, at least to some extent, reversible, an equilibrium is set up in which the ratio of [C*]/[C*]+[C*-R] is determined by the amount of the unlabelled compound C which is present. If C* is separated from C*-R and the level of activity of each separated part measured, then the value of the ratio is easily calculated. The amount of the unlabelled compound C can then be determined, in relative or absolute terms, by the use of standard preparation of compound C, to generate a calibration curve.
The technique is described, with examples, in a Review Paper by R. S. Yalow and S. A. Berson in IAEA-SM-124/106, pages 455-481.
Separation of C* from C*-R is generally effected in two stages. In the first stage, either C* or C*-R is caused to change phase for example by being precipitated from solution, or adsorbed on to an inert carrier, or passed into a water-immiscible phase. In the second stage, the two phases are removed from one another, e.g. by filtration, or more usually, by centrifuging followed by decanting the supernatant liquor. With the two phases separated into different vessels, it is a simple matter to determine the level of activity in each.
THE SEPARATION OF THE TWO PHASES BY REMOVAL OF ONE FROM THE REACTION TUBE INTO ANOTHER VESSEL IS TIME-CONSUMING AND GIVES RISE TO INACCURACIES. In particular, the transfer of material from one vessel to another is rarely complete.
An experiment is normally performed using a number of tubes, each containing the same amount of C* and R, of which tubes contain an unknown amount of the compound C to be assayed, while other tubes contain a known amount of the compound C and are used as standards to generate a calibration curve. The total volume of material in each tube used in an experiment is generally the same. In these circumstances another known procedure is sometimes used.
According to this known procedure, only a part of a liquid phase is withdrawn, e.g. by pipetting, from the reaction vessel, and an accurately measured volume thereof is placed in another vessel, and its level of radioactivity determined. The assumption is made that the accurately measured volume of liquid represents a constant proportion (as between different tubes in the same experiment) of the total volume of material. As before, the transfer of an accurately measured volume of radioactive liquid from one vessel to another is time-consuming and gives rise to inaccuracies.